Amino-deoxy-disaccharides and amino-deoxy-oligosaccharides

ABSTRACT

Synthesis of an amino-disaccharide, amino-oligosaccharide or a derivative thereof, characterized in that a monosaccharide, a disaccharide, an oligosaccharide, a glycoside or a derivative thereof, in the presence of a glycosidase as catalyst, is reacted with an amino-deoxy-saccharide or a derivative thereof, and that the amino-saccharide is isolated from the product mixture directly or after chemical/enzymatic modification.

REFERENCE TO A RELATED APPLICATION

This is a continuation of international application PCT/SE94/00461,filed May 17, 1994, which designated the United States, and which isincorporated by reference in its entirety.

INTRODUCTION AND BACKGROUND

The present invention describes a new method for synthesis of anamino-deoxy-disaccharide or an amino-deoxy-oligosaccharide.

It has been found that the oligosaccharide part of variousglycoconjugates (especially glycolipids and glycoproteins) have a numberof important functions in vivo (Biology of Carbohydrates, vol. 2,Ginsburg et al., Wiley, N.Y., 1984; The Glycoconjugates, vol. I-V,Academic Press, New York; S. Hakomori, Ann. Rev. Biochem., vol 50, pp.733-64; Feizi, Nature, pp 314, 1985; S. Hakomori, Chemistry and Physicsof Lipids, vol. 42, pages 209-33). Among other thing it was found that

the carbohydrate structures are important for the stability, activity,localization, immunogenicity and degradation of glycoproteins;

carbohydrates are antigenic determinants (for example blood groupantigens);

carbohydrates function as receptors when bound to cell surfaces forpathogens, proteins, hormones, toxins and during cell--cellinteractions;

carbohydrates are important for oncogenesis, since specificoligosaccharides have been found to be cancer-associated antigenicdeterminants;

frequently, only a smaller sequence (di- or trisaccharide) of thecarbohydrate part of the glycoconjugate is required for full biologicalactivity (e.g. receptor activity).

Universities and industry are at present working intensely on developingthe use of biologically active oligosaccharides within a number ofdifferent fields, such as

novel diagnostics and blood typing reagents;

highly specific materials for affinity chromatography;

cell specific agglutination reagents;

targetting of drugs;

monoclonal antibodies, specific against e.g. cancer-associated reagents;

therapy;

development of a new type of therapy, as an alternative to antibiotics,based on the inhibition of the attachement of bacteria and virus on cellsurfaces with specific oligosaccharides;

stimulation of the growth of plants and protection against pathogens.

Besides the above mentioned areas, a considerable future market isenvisaged for fine chemicals based on biologically active carbohydrates.

Amino-saccharides, where an --OH group in the saccharide is exchangedfor an --NH₂ group, in several cases have a higher (or modified)biological activity than the corresponding hydroxyl- orN-acetylamino-deoxy-saccharides, e.g. in the binding to selectinsimportant for the initiation of inflammation processes (binding ofleucocytes to epithelial cells in blood vessels). The opportunity to usesuch saccharides therapeutically, e.g. in acute or chronic inflammatoryconditions (e.g. reperfusion, injury, and septic shock) is investigated.An important component in this and in other cases is the selectivesynthesis of di- and oligosaccharides in sufficient quantities. Thepresent invention describes a novel technique for synthesis ofamino-saccharides.

Amino-deoxy-di-, tri- or higher oligosaccharides which contain one ormore amino --NH₂ groups are of high interest for food, agricultural,pharmaceutical or diagnostic applications of carbohydrates, to modifythe metabolism of the substance and/or to increase the biological effectof the natural substance.

About ten different monosaccharides are included in the carbohydratepart of the glycoconjugates: D-glucose (Glc), D-galactose (Gal),N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-neuraminic acid (Neu5Ac),D-mannose (Man), L-fucose (Fuc), N-acetyl-D-galactosamine (GalNAc),xylose (Xyl), and arabinose (Ara) (the abbreviations in brackets areaccording to IUPAC-IUB's abridged terminology for monosaccharides,J.Biol.Chem. (1982), vol. 257, pages 3347-3354, in which publication onealso can find the nomenclature used in this text to describeoligosaccharide sequences). The number of possible structures will bealmost infinitely great because both the anomeric configuration and theposition of the O-glycosidic bond can be varied.

The organic chemical techniques used today for synthesis of theseoligosaccharide structures require an extensive protective groupchemistry with many steps of synthesis and expensive catalysts (see e.g.Binkley: Modern Carbohydrate Chemistry, Marcel Dekker, New York, 1988,with references). Low total yields are obtained in these complicatedreaction schemes and the technique is not favorable, especially forlarger scale work.

Selective chemical synthesis of amino group containing carbohydrates andderivatives require advanced protection group chemistry with manysynthetic steps. (see e.g. Binkley: Modern Carbohydrate Chemistry,Marcel Dekker, New York, 1988, with references). Efficient techniquesfor preparation of such carbohydrates and derivatives thereof are thusdesired.

The present invention describes a process which makes possible adrastically simplified synthesis of derivatised or unmodified di-, tri-,and higher oligosaccharides which contain at least one --NH₂ (amino)group. Carbohydrate amino derivatives which required several reactionsteps to synthesis with previous methods, can, with the method accordingto the present invention, now be obtained with only one reaction stepand with absolute stereospecificity.

Enzymes are nature's own catalysts with many attractive characteristics,such as higer stereo-, regio-, and substrate selectivity as well as highcatalytic activity under mild conditions. Today, great hopes aretherefore placed in being able to utilize enzymes for large-scaleselective synthesis of oligosaccharides with fewer reaction steps andconsequently higher total yields than by organic chemical methodology.

Both hydrolases (glycosidases, EC 3.2) and glycosyltranferases (EC 2.4)can be used for synthesis (glycosidases: see Nisizawa et al, in "TheCarbohydrates, Chemistry and Biochemistry", 2nd Ed., vol. IIA, pages242-290, Academic Press, New York, 1970). With glycosidases, reversedhydrolysis (equilibrium reaction) or tranglycosylation (kineticreaction) are often used to obtain synthesis (see e.g. K. G. I. Nilsson,Carbohydr. Res. (1987), vol. 167, pages 95-103; Trends in Biochemistry(1988), vol. 6, pages 256-264). ##STR1## (DOH is donor saccharide, DORis donor glycoside with α- or β-glycosidically bound aglycon (--R), HOAis acceptor saccharide and EH is enzyme).

With transferases, a nucleotide sugar (non-limiting examples areUDP-Gal, CMP-Sia, UDP-GalNAc, GDP-Fuc, etc), which is relativelyexpensive, is used as donor. Furthermore, glycosidases are abundant andcan often be used directly without purification.

The synthetic method according to the invention includes at least oneprocess characterized by that a glycosidase (EC 3.2) is used to catalyzean equilibrium or a transglycosylation reaction between an acceptorsubstance, which consists of a mono-, di-, tri- or higheroligosaccharide which contains at least one amino-deoxy-group ##STR2##and which is modified or unmodified, and a glycosyl donor, which is amonsaccharide, disaccharide, oligosaccharide or a glycoside orderivative thereof, and that the product is used for continued synthesisand/or is isolated from the product mixture.

In this way one obtains, according to the invention, stereospecificsynthesis of di-, tri-, or higher amino-deoxy-oligosaccharides orderivatives thereof, which can be used directly, or after furthersynthesis, for a number of various applications, e.g. forpharmaceutical/medical/diagnostical studies, for applications in therapyor diagnostics, as additives in cosmetics or in food, for modificationof separation material, affinity chromatography, modification of aminoacids, peptides, proteins, fatty acids, lipids, enzymes, or recombinantproteins.

In the synthesis according to the invention, the capacity ofglycosidases to form stereospecific glycosidic linkages between aglycosyl donor (DR in the scheme below, where D symbolizes thetransferred carbohydrate part) and a glycosyl acceptor (HOA), summarizedin the scheme below: ##EQU1## The reaction according to the inventioncan be carried out according to two principles, either with equilibriumcontrolled synthesis (R═H), or with transglycosylation reaction (R═F, oran organic group; kinetically controlled reaction). These general typesof reactions are well know to the expert and their carrying out, as wellas the choice of glycosyl donor and glycosidase, do not restrict thescope of the invention.

SUMMARY OF THE INVENTION

Synthesis of an amino-disaccharide, amino-oligosaccharide or aderivative thereof, characterized in that a monosaccharide, adisaccharide, an oligosaccharide, a glycoside or a derivative thereof,in the presence of a glycosidase as catalyst, is reacted with anamino-deoxy-saccharide or a derivative thereof, and that theamino-saccharide is isolated from the product mixture directly or afterchemical/enzymatic modification.

DETAILED DESCRIPTION OF THE INVENTION

The synthesis, according to the invention, is carried out by reacting amonosaccharide, a disaccharide, an oligosaccharide, a glycoside or aderivative thereof with an amino-deoxy-saccharide or a derivativethereof in the presence of a glycosidase (EC 3.2) as a catalyst.

As nonlimiting examples of amino-deoxy-monosaccharides which can be usedas acceptors one can mention a 2-amino-2-deoxy-glucopyranoside, a2-amino-2-deoxy-galactopyranoside, or a 2-amino-2-deoxy-mannopyranoside(thus, in the scheme below, R₃, R₄ and R₆ are --OH and R₁ is one of e.g.pentenyl-, --SEt, --SPh, --OEtBr, --OEtSiMe₃, --OAll, --OPh, --OCH₂ Ph,or --OR, where R is e.g. CH₃ (CH₂)n; n is an integer, preferably in therange 0-12; or where R is for example an amino acid residue, peptideresidue, or a derivative thereof): ##STR3##

Other nonlimiting examples of amino-deoxy-saccharides is an 2, 3, 4, 5or 6 amino-monosaccharide as above, which has been derivatised in one ortwo of the positions 2, 3, 4, 5 or 6. As examples of such derivativesone can mention derivatives in which one or two of the hydroxyl groupshave been modified to an allyloxy- (CH₂ ═CH--CH₂ O--), bensyloxy- (PhCH₂O--), bensoyloxy-(PhCOO--), chloroacetyloxy-(ClCH₂ COO),p-methoxybensyloxy-(p-MeO-PhCH₂ O--), trityl- (Ph₃ CO--),trialkylsilyloxy-, tosylate-, mesylate-, phosphate-, sulfate-,carboxylate, esters such as RCOO-- where R is CH₃ (CH₂)n (n=1-20) or apivaloyloxy-group or derivatives in which two vicinal hydroxyl groupshave been modified e.g. bensylidene acetal, isopropylidene ketal or anortho ester, pivaloyl-group, tetrahydropyranyl,(2-methoxyethoxy)methylisopropylidene ketal, cyclohexylidene ketal,benzylidene acetal, orthoester, --ONO₃, derivative of sulfate-,phosphate-, carboxylate, esters i.e. of the type --OC(O)R as acetyl-,butanoyl-, octanoyl-, benzoyl-, pivaloyl-, etc. The structures below,modified in a similar way, can also be used as acceptor substances inthe method according to the invention.

If modified amino monosaccharide is used, the choice of the type ofmodification of the acceptor is decided by what is desired in thespecific situation and the literature is rich in information onprotection groups/modification of carbohydrates and carbohydratesynthesis in general (e.g. "Modern Carbohydrate Chemistry", Binkley,Marcel Dekker, 1988 with references; Paulsen, Chem. Soc. Rev., vol. 13,pages 15-45). Below are a few examples of acceptor substance categorieswhich can be used according to the invention but which in no way aremeant to restrict the scope of the invention.

Similarly, modified amino di, tri- or higher oligosaccharides can alsobe used as acceptors. ##STR4##

In the structures I-XI above, R₃ is for example an alkyl, allyl, benzyl,chlorobensyl, benzoyl-group or another type of suitable protection groupfor the specific synthesis. R₆ can be aromatic group such as Ph- or analkyl group (e.g. propyl- or (CH₃)₃ -group). In the structures XII-XVII,R₃ is for example an acetyl-, phenoxyacetyl-, methoxyacetyl- or anchlorometoxyacetyl group. R₆ can be an aromatic group, such as Ph- or analkyl group (e.g. propyl- or (CH₃)₃ group). If R₂ for example is H, thenR₁ is one of the groups which has been mentioned for R₁ above, and viceversa if R₁ instead is H. Similarly, position 4 can be modified insteadof position 3 or 6 in the examples above, and other positions than the 2position may be modified with an amino-deoxy group.

As an example to illustrate the invention, but which in no way is meantto limit the scope of the invention, can be mentioned that if, forexample, α-galactosidase is used as enzyme and 2-amino-2-deoxyα-D-galactopyranoside is used as acceptor substance, and if, for exampleraffinose, methyl α-D-galactopyranoside, GalαF (F=fluoro) (orp-nitrophenyl) α-D-galactopyranoside is used as glycosyl donor(transglycosylation reaction), an α-glycosidically linked2-amino-2-deoxy-digalatosyl derivative of the type ##STR5## i.e. a 2-NH₂-2-deoxy-derivative of Galα1-3Galα-R, is obtained. As another example,if I is used as acceptor and a α-galactosaminidase, and e.g.(GalNAcα-OPh, GalNAcαF or GalNAcα-OPhNo₂ -p, is used as glycosyl donor,a 2-O-derivative of GalNAcα1-3Galα-R is obtained.

The products can be used if desired for further synthesis, e.g. ofhigher oligosaccharide with chemical synthesis and the literature isextensive on how to use such partially protected carbohydrates (seereferences in Binkley and Paulsen mentioned above).

If a β-galactosidase is used instead of an α-galactosidase and iflactose, or for example p-nitrophenyl-β-D-galactopyranoside, is used asglycosyl donor, and if 2-amino-2-deoxy-glucose or a derivative thereof(see e.g. XII-XVII above) is used as acceptor, β-bound derivatives ofGal-GlcNH₂ or Gal-GlcNH₂ --R are obtained. Examples of partiallyprotected Gal-GlcNH₂ or Gal-GlcNH₂ --R derivatives, which can be usede.g. for synthesis of Lewis-x or Lewis-a trisaccharide structures (orwhich can be used for further synthesis of disaccharide derivatives ofthese) are given below: ##STR6## Moreover, if instead an α-L-fucosidaseis used with, for example, nitrophenyl α-L-fucopyranoside or with Fucα-Fas glycosyl donor, one can synthesis the corresponding derivatives ofe.g. α-bound Fuc-Gal-NH₂ --R and of α-bound Fuc-GlcNH₂ --R with themethod according to the invention, similarly withN-acetyl-β-glucosaminidase or N-acetyl-β-galactosaminidase one canprepare derivatives of β-bound GlcNAc-Gal-NH₂ and GlcNAc-GlcNH₂ orGalNAc-Gal-NH₂ and GalNAc-GlcNH₂, respectively, with β-glycosides ofGlcNAc and GalNAc, respectively, as glycosyl donors. Similarly,α-sialidase can be used to catalyze synthesis of e.g. sialylated2-amino-2-deoxy-galactose (Neu5Acα-GalNH₂) or of2-amino-2-deoxy-galactosamine-derivatives (derivatives ofNeu5Acα-GalNH₂) by employing e.g. nitrophenyl glycoside ofN-acetylneuraminic acid and a partially protected2-amino-2-deoxy-galactose derivative, respectively, as acceptor.

If an endoglycosidase is used, one can prepare longer oligosaccharidederivatives with the method according to the invention. Then, the donorsubstance is of the type disaccharide, tri- or higher oligosaccharide ora glycoside, e.g. nitrophenyl glycoside of any of these. Similarly, anyof the R groups of the acceptor substance might be a saccharide unit.

The reaction above can also be carried out as equilibrium reactions withmonosaccharides as glycosyl donors.

The benzyl- or the allyl group (or other groups mentioned in connectionwith the figures above) in the products above, can easily be chemicallychanged by the expert to a wide range of groups, and in this wayselective synthesis of different amino-deoxy-disaccharide derivatives(e.g. O-phosphate, O-sulfate, etc) or higheramino-deoxy-oligosaccharides can be selectively synthesized according tothe invention.

The substrates are selected with regard to the oligosaccharide which isto be synthesized, and are often commercially available or can bysynthesized by organic or enzymatic methods and therefore do notrestrict the use of the invention. The donor substrates which are usedaccording to the invention are of the same type which have been used inprevious transglycosylation reactions (see for example the articles byK. G. I. Nilsson in Carbohydrate Res. vol. 167 and in Trends inBiotechnology, vol. 6 as noted above).

As further examples of acceptor substances which can be used with themethod according to the invention can be mentioned amino-deoxy di- oroligosaccharides (or glycosides thereof) in which the carbohydrate partcontains one or more of the following monosaccharides: D-glucose,D-galactose, D-mannose, N-acetyl-neuraminic acid,N-acetyl-D-galactosamine, N-acetyl-D-glucosamine and L-fucose, oranalogs of these. When the acceptor substance is a glycoside, theaglycone can be a glycosidically bound (α- or β-configuration) aliphaticor aromatic compound (as for example methyl, ethyl, 2-bromoethyl,(CH₂)_(n) COOMe, n>1, allyl or other substances that can be polymerized,benzyl, pentenyl, trimethylsilylethyl, amino acids, derivatives thereof,peptides, derivatives thereof, nitrophenyl, etc).

Other types of aglycons of special interest are amino acids (serine,threonine, hydroxyproline, hydroxylysine, asparagine, etc), peptides,lipids and derivatives or analogs to substances within these threegroups. The amino acid and peptide glycosides can be protected on theiramino and/or carboxyl groups with the common protecting groups used inpeptide synthesis (FMOC, CBZ, BOC, etc). By using usch aglyconesfragments or analogs of glycoconjugates can be synthesized according tothe invention; the terms aglycones, fragments and analogs are terms wellknown to those skilled in the art. Moveover, the aglycon can be anamino, nitrile, or an amido group or a fluorogenic substance, or maycontain a phosphate, sulfate, or carboxyl group or a derivative thereof.Another important type of amino-deoxy saccharide derivatives consists ofsubstances where the ring oxygen (i.e. the C-5 oxygen of hexoses), hasbeen replaced by sulfur, nitrogen, etc. The glucose analog moranoline,where the C-5 oxygen has been replaced by nitrogen, is an example ofsuch a derivative. Oligosaccharide analogs that are efficient inhibitorsagainst enzymes or carbohydrate binding proteins may in this manner besynthesized according to the invention.

The donor substances which can be used with the method according to theinvention are the same as those employed in previous methods involvingenzymatic transglycosylations (see references above) and thus do notlimit the scope of the invention.

As examples of donor substances that can be used with the methodaccording to the invention may be mentioned monosaccharide glycosidesand di- or oligosaccharides (or gylcosides thereof) in which thecarbohydrate part contains one or more of the monosaccharidesD-galactose, D-glucose, D-mannose, N-acetyl-neuraminic acid,N-acetyl-D-galactosamin, N-acetyl-D-glucosamin and L-fucose. As examplesof suitable glycosyl donors may be mentioned the nitrophenyl α- orβ-glycosides of the monosaccharides above, lactose, dimannose andraffinose. As examples of suitable donor substances for endoglycosidasesmay be mentioned nitrophenyl derivatives of biologically activecarbohydrate sequences (e.g. Galβ1-3GlcNAcβ-OPhNO₂ -p), biologicallyactive oligosaccharides or structures of the type Glc(β1-3Glc)_(n)β1-3Glc (n>1).

The concentration of the glycosyl donor in the reaction mixture isselected with regard to the oligosaccharide which is to be synthesizedand also with regard to the properties of the enzyme and therefore donot restrict the use of the invention. In some cases, addition of thedonor in smaller portions may be advantageous in order to minimize therisk that the donor also acts as an acceptor (unless this is desired).

The enzymes are selected primarily with regard to which oligosaccharideis to be synthesized. The enzyme may be used in situ or after partial orcomplete purification from their natural environment. The enzyme may beused in soluble form or immobilized to a solid support by e.g.adsorption, encapsulation, chelation, precipitation or covalent binding.

Examples of α- and β-glycosidases which may be used according to theinvention are D-mannosidases, D-galactosidases, L-fucosidases,N-acetyl-D-galactosaminidases, sialidases, hexosaminidases and otherglycosidases of EC group 3.2 (Enzyme Nonmenelature, Academic Press,1984). Both endo- and exoglycosidases may be used in the methodaccording to the invention.

The degree of purity of the enzyme employed is not critical. The enzymemay be used in situ or after complete or partial isolation from itnatural biological environment. Also, a crude extract of the organism ora tissue thereof may be used. The enzyme may also have been obtainedafter precipitation with e.g. ammonium sulfate. The enzyme may bepresent in crystalline form or be enclosed within micelles. Thebiochemical literature is rich in detailed information about thepurification and isolation of glycosidases. The enzyme may be producedwith recombinant techniques. Then, if desired, one or more of the aminoacids in the amino acid sequence of the enzyme may be changed in orderto optimize the properties of the enzyme, e.g. themostability, catalyticefficiency and/or regioselectivity.

The enzyme may be used in soluble form or may be immobilized by e.g.adsorption, encapsulation, chelation, precipitation or covalent bindingto a solid support, such as a polymeric substance, or a derivativethereof which is insoluble in protic or aprotic solvents (Methods inEnzymology, vol. 44, Academic Press, 1976). The form selected is notcritical to the invention. If the enzyme is used in soluble form, it mayfirst have been chemically modified in a suitable manner in order toe.g. increase the thermostability or the stability in organiccosolvents. Enzyme immobilized to an insoluble polymer comprising, forexample, agarose, cellulose, hydroxyethyl acrylate, glass, silica,polyacrylic amide, polyacrylate-based plastics, etc., is readilyseparated from the product mixture, and the enzyme may thus be reused.An additional advantage is that in many cases a certain stabilizationagainst elevated temperatures and organic cosolvents is obtained.

Moreover, the products can be used for further enzymatic synthesis withglycosidases or glycosyltranferases. For example, α-sialyltranserase canbe used to catalyze the formation of sialylated Gal-GlcNAc-derivativesand β-galactosyltransferase can be used to form oligosaccharidederivatives of the type Gal-GlcNAc-Gal-R, which then can eventually besialylated and/or be used for further chemical synthesis, etc.

If a modified 2-amino galactoside or glucoside is used as acceptor, thechoice of aglycon is made with regard to the application of the product.Aglycons of special interest are amino acids (serine, threonine,hydroxyproline, hydroxylysine, asparagine, etc.) peptides, lipids andderivatives or analogs of substances within these three groups. Aminoacid or peptide glycosides can be protected on their amino- and/orcarboxyl functions with common groups used in peptide synthesis (FMOC,CBZ, BOC, etc). Product obtained with modified alkyl glycosides (e.g.modified methyl-, octyl-, docecyl glycosides) as acceptor substances,may be used as inhibitors in affinity chromatography or in agglutinationtests, inhibition-based therapy or for drug-targeting, as structuralunits for further enzymatic synthesis. Nitrophenyl glycosides can bereduced to aminophenyl glycosides. Glycosides with a polymerisableaglycon, as for example 2-hydroxyethylmethacrylate, can be used. As anexample of a N-glucosidically bonded aglycon, --NHCO (CH₂)₅ NH₂, may bementioned. Other types of aglycons which can be used are those used e.g.in the synthesis of glycolipids/analogs for conversion toceramides/analogs, e.g. aglycons of the type described by Magnusson etal in J. Org. Chem., 1990. Thioglycosides (e.g. SEt or SPh) can be usedwith the method according to the invention to produce products which aresuitable for further chemical synthesis. The choice of protectiongroup/derivative, aglycon, position of derivatized hydroxyl groups, canbe used to influence the yield and regioselectivity of the reactionswith the method according to the invention. Thus, for example, the useof more hydrophobic aglycons (e.g. p-metoxy-benzyl-, benzyl-, comparedwith e.g. allyl-) can result in a higher yield at the same acceptorconcentration.

The enzymes are selected with regard to the final oligosaccharide whichis to be synthesized. The enzyme can be used in situ (especially severalglycosidases) or after partial or complete purification from theirnatural environment. The enzyme may be used in soluble form orimmobilized to a solid phase by e.g. adsorption, encapsulation,chelation, precipitation or covalent binding. Simultaneous use ofglycosidase and glycosyltransferase in soluble form or immobilized to asolid phase (eventually co-immobilized) may be advantageous according tothe invention in facilitating the conversion of the intermediateoligosaccharide product to the final product oligosaccharide. In thisway the method according to the invention gives important advantagescompared to previous methods: purification of intermediary product isnot necessary, secondary hydrolysis is minimized (i.e. higher yield),and trisaccharides or higher oligosaccharides can be synthesized in aminimum of "pots" (in some cases one-pot reactions). This is facilitatedby the high acceptor specificity of most glycosyltransferases: thetransferase does not react with the wrong isomer.

The synthetic procedure according to the invention can be carried outunder highly diverse conditions as regards, for example, pH, type ofbuffer, temperature and concentration of the reactants. Variouscosolvents (N,N-dimethyl formamide, acetonitrile, dimethyl sulfoxide,dioxane, pyridine, methanol, ethanol, ethylene glycol, etc) may be usedand in varying concentrations together with water (0-99%). Moreover, thereactions can be carried out in two-phase systems: water-organicsolvent. THe use of acceptor aminosaccharides modified with organicgroups facilitates recovery of the product in the organic phase.

The reaction conditions are not critical but are selected primarily onthe basis of the properties of the reactants employed in the synthesisconcerned, and also on the basis of practicality. For example, it may bementioned that it is usually convenient to use room temperature withenzymes and, in the case of water-rich medium, the pH is usually in therange 4-11. The solubility of amino-saccharides in water isincreased/decreased by decreased/increased pH, and in some cases a pHbelow 8 and above 4 is preferably used to increase the solubility of theacceptor amino-saccharide.

Organic cosolvents may be used to minimize the hydrolytic side-reaction.For the same reason, two-phase systems may be used. Examples ofcosolvents are tetrahydrofurane, acetonitrile, DMF. The choice ofsolvent and of the concentration or organic solvent can easily be madeby the expert and does not limit the scope of the invention. Use of highconcentrations of organic solvent (up to almost 100% of the total volumesolvent) can be especially advantageous when acceptor derivatives withhydrophobic groups which have good solubility in organic solvents areused, e.g. acceptors modified with ester groups (e.g. acetyl-, bensoly-,butanoyl-, pivaloyl-, octanoyl-grupper, etc.) and/or with for exampleallyl-, bensyl-, trityl- or other groups. In this way relatively highconcentration of the acceptor can be achieved in organic solvents andthe hydrolytic side-reaction can be decreased due to the low watercontent. The method according to the invention allows synthesis inorganic solvent of e.g. amino deoxy trisaccharde derivatives and higheroligosaccharide derivatives with exoglycosidases by using hydrophobicprotected derivatives of amino deoxy di-, tri- or oligosaccharides,which has only one or a few free dydroxyl groups, as acceptors.

To increase the solubility/availability in organic solvent andfacilitate the reaction with the donor substance, one can use forexample phenyl boronate, which forms a complex with saccharides withvicinal diols and the resulting donor-boronate complex has, because ofthe phenyl group, a higher solubility in organic solvent.

The reaction temperature may also be varied to influence product yieldand the stability of the enzyme and does not restrict the scope of theinvention. The temperatures most frequently used lie in the range 4°-55°C., but lower temperatures and temperatures below 0° C. can be usedwhich can be facilitated if organic cosolvent is used. Highertemperatures can be used with thermostable glycosidases and substrates,and also with enzymes stabilized against thermal denaturation byemploying, for example, high substrate concentrations (Johansson et al,Biotechnol. Lett. (1986), vol. 8, pages 421-424). An advantage with hightemperatures is, for example, that high substrate concentrations may beused, which reduces the water activity and thus increases the yield ofproduct. Another advantage is that the activity of the enzyme increases,which means shorter reaction times at increased temperatures. Oneadditional advantage is that glycosides, e.g. methyl or ethylglycosides, which are hydrolyzed slowly at room temperature can be usedas suitable gylcosyl donors at increased temperatures (50°-60° C.). Theupper temperature limit is determined by the thermostability of theenzyme in the reaction medium. For some transglycosidations, a lowertemperature was found to give a higher yield of product glycoside.

The concentration of the acceptor is a parameter which can be used toinfluence the yield of the reactions according to invention. Highconcentrations are preferrable in both equilibrium andtransglycosylation reactions to mimimize hydrolytic side-reactions,which usually means that depending on the solulility of the acceptor, ca0.05-7 M concentration of acceptor is used. A high concentration ofdonor is often used and especially in equilibrium reactions. In general,high concentrations of substrates are obtained by heating the reactionmixture to near the boiling point for a few minutes, allowing thesolution to cool to the reaction temperature (usually 4°-75° C.,depending on the temperature for optimum yield and thermostability ofthe enzyme/substrate) and then add the enzyme. Cosolvents can be used toincrease the solubility of substrates with hydrophobic groups.

The reaction can be monitored by means of TLC, HPLC, or byspectrophotometric measurement of liberated aglycon (e.g. p-nitrophenol,400 nm). Charring of TLC-plates with ninhydrin may be used for detectionof NH₂ -groups. When a desirable yield of the product has been obtained,the reaction is terminated by denaturation of the enzyme by changing thepH, increasing the temperature and/or adding organic cosolvent (such asethanol). Heating to 60°-85° C. for 3-5 min (eventually followed byaddition of ethanol to a concentration of about 80%) is usuallysufficient.

Various techniques may be used for isolation of the product.Precipitation from the water-phase or from an organic solvent (such ase.g. ethanol, methanol, ethyl acetate) is useful, especially when anexcess of one of the reactants is used or when the donor, acceptor orproducts have different solubilities. After the equilibrium controlledsynthesis or the transglycosylation reaction and after e.g. heattreatment as above and dilution of the reaction mixture, it can beuseful to add a second glycosidase, which has a differentregioselectivity than the glycosidase used in the synthesis. In thisway, any unwanted regioisomers (for example with 1-6 linkages) may bemore or less selectively hydrolyzed, which facilitates isolation of thedesired product.

Precipitation, extraction of the water phase with an organic solvent,and hydrolysis of byproducts are complementary to chromatography (ionexchange chromatography, gel filtration, HPLC with, for example,amino-silica, reversed phase silica or the new Dionex columns).

Some examples of how the invnetion can be used in practice, but which byno means are meant to restrict the scope of the invention, are givenbelow.

Examples of substances, which can be used as donor saccharides (DR,where D is the transferred glycosyl group in the reaction) according tothe invention is D-glucose, D-mannose, L-fucose, D-galactose, xylose,N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetyl-neuraminicacid, glycosides of these, disaccharides or oligosaccharides containingone or more of the monosaccharides above (e.g. lactose, raffinose,chitobiose), and derivatives of any of the substances mentioned above,e.g. modified in one or more of the ring hydroxyl groups.

The reaction according to the invention can therefore be summarized asfollows: ##EQU2## where D is glucosidically bound to the saccharide unitof the amino-saccharide. Endo- or exoglycosidase (EC group 3.2) are usedas enzyme, and the reaction is carried out as a transglycosylationreaction. The equilibrium type reaction may also be chosen Non-limitedexamples of exoglycosidases are α-galactosidase, β-galactosidase,β-N-acetyl-glucosaminidase, β-N-acetyl-galactosaminidase,α-L-fucosidase, α-sialidase, α- or β-xylosidase, α-mannosidase orβ-mannosidase.

The reaction conditions are chosen according to the reaction; somenon-limiting examples are given below: The concentration of reactantsare usually in the interval 0.05 M to above 1 M depending on thesolubility of the reactants, the temperature is usually in the range 0°to 80° C. and the reaction is usually carried out in buffered water, pH4-9; the pH and temperature are chosen according to e.g. the enzyme'sproperties, eventually an organic co-solvent can be used (1-99% of e.g.tetrahydrofurane or acetonitrile). The reaction is usually stopped whenthe maximum yield of amino-saccharide product has been obtained and theproduct is isolated with, for example, one or more of columnchromatography (adsorbent for example ion-exchange material, Sephadex orsilica), extraction, precipitation, crystallization and/or filtrationtechniques.

EXAMPLES

As a non-limiting specific example one can mention the production ofthioetylβ-D-galactopyranosyl-(6-bensyl-2-amino-2-deoxy)-β-D-glucopyranosideproduced via reaction between nitrophenyl β-D-galactopyranoside andthioetyl (6-bensyl-2-amino-2-deoxy)-β-D-glucopyranoside in e.g. sodiumacetate buffer, pH 5, catalysed by β-galactosidase.

The product can be used either directly e.g. in biological/medicalapplications or can be used as a synthetic intermediate for furthersynthesis of higher oligosaccharides or other derivatives.

Synthesis of derivatives of Galβ1-3GlcNH₂ and Galβ1-4GlcNH₂ respectively(constituents of Lewis-blood group substances, such as Lewis-a, Lewis-xand sialylated structures): By using for example derivatised glycosideof glucosamine, such as e.g. structures IX or II, as acceptor dissolvedin for example (1/1 V/V) tetrahydrofurane:sodium acetate buffer (pH 5.5,0.05 M), Galβ-OPpNO₂ -O as donor, and β-galactosidase as catalyst,structures of the types below can be obtained: ##STR7## Such structurescan be used directly in various applications, or can be used for furtherchemical or enzymatic synthesis. The galactosyl moiety can for examplebe modified with chemical or enzymatic methods (lipase or galactoseoxidase, followed by chemical modification).leaving one free hydroxylgroup in the glucosaminyl-moiety, which can be modified with for examplea fucosyl group.

Similarly, by using an acceptor of the type below, the correspondingβ-bound 3-O-protected Gal-GlcNH₂ -derivative can be obtained. ##STR8##

After protection of the free hydroxyl groups and the amino group in theproduct and deprotection of the 3-O-position can, for example, anα-bound L-fucosyl group can be introduced, which gives the modifiedLewis-x structure, which can be, for example, sialylated to give e.g.NeuAcα2-3Galβ1-4(Fucα1-3)GlcNR₂ --R. In an analogous way, one canproduce regioisomers, such as Galβ1-3(Fucα1-4)GlcNR₂ --R, andanalogs/derivatives of Lewis-x, Lewis-a, and of sialylatedLewis-substances.

EXAMPLE 1

A non-limiting example of the application of the method according to theinvention is the synthesis of Galβ1-3(6-O-Bn)GlcNH₂ βSEt employingthioethyl (6-O-bensyl-2-amino-2-deoxy)-β-D-glucopyranosid, abbreviated(6-O-Bn)GlcNH₂ βSEt, as acceptor and galactose or lactose or agalactoside, e.g. nitrophenyl α-D-galactopyranoside as glycosyl donorand β-galactosidase from ox testes as catalyst.

Other sources of β-galactosidase which gives the linkage may be usedaccording to the invention. The reaction was carried out at roomtemperature with initial concentration of substrates typically in therange of 0.06 M to 0.3 M. The donor was used in excess over theacceptor. A crude ammonium sulfate precipitate of the enzyme was used inthe reaction, which was carried out at pH 5 in 0.05 M sodium acetatebuffer. The reaction was terminated by heat treatment for ca 5 minutesin a boiling water bath. The product was isolated by e.g. adjusting thepH to ca 10.5 (minimizing the charge on the amino group), extraction ofthe water phase with ethyl acetate, followed by butanol extraction, thebutanol phase was evaporated and the residue dissolved in water andapplied to an ion-exchanger (in this example a sulphopropyl groupcontaining fast-flow ion-exchanger from Pharmacia). The fractionscontaining the product was evaporated and the product dried and analyzedby NMR.

Similarly, another 6-O-substituted product than the bensyl-substitutedproduct and/or another type of 1-substituted derivative than the1-thioethyl substituted product, can be obtained by instead of(6-OBn)GlcNH₂ βSEt employing another 6-O- and/or 1-substituted acceptoras exemplified in the description.

Another non-limiting example is the synthesis of Fucα1-4(6-O-Bn)GlcNH₂βSEt using thioethyl (6-O-bensyl-2-amino-2-deoxy)-β-D-glucopyranoside,abbreviated (6-O-Bn)GlcNH₂ βSEt, as acceptor and fucose or afucopyranoside, e.g. nitrophenyl α-L-fucopyranoside as glycosyl donorand α-L-fucosidase from ox kidney as catalyst.

The reactions above can for example be carried out with ca 0.1 Mconcentrations of substrate and the isolation can be carried out by theuse of an ion changer (e.g. sulphopropyl-containing material) andextraction of the water phase with a suitable solvent, e.g. butanol orethylacetate.

The two substances above are of interest for example asinhibitors/modifiers of selectin-carbohydrate interactions in vivo suchas in different inflammatory reactions e.g. septic chock, rheumatism andasthma, but also as inhibitors/modifiers of the up-regulation ofIgE-synthesis in vivo (for example inhibition, modification of theFceRll-CR interaction, see e.g. Nature (1993), volume 366, page 41-48,and references therein, for an overview).

One of the advantages with the method according to the invention, isthat the amino-disaccharide- or the amino-oligosaccharide product andderivatives thereof can be synthesized directly, and thus nomodification of the amino-group is required after theglycosidase-catalysed reaction. Another advantage is that partiallymodified amino-sugar derivatives can be produced stereospecifically andunder reaction specific conditions. Such derivatives can be useddirectly in various applications or as synthetic intermediates forfurther synthesis of higher oligosaccharides or other derivatives.

EXAMPLE 2

Synthesis of Galβ1-4(6-OBn)GlcNH₂ βSEt. The synthesis of this compoundis achieved similarly as above, but another source of enzyme which givesthe β1-4 linked product, is employed, e.g. a yeast enzyme such as theone from Bullera singularis. In this case the reaction can be carriedout as a fermentation with e.g. lactose as the glycosyl donor and withintact cells.

Similarly, another 6-O-substituted product than the bensyl-substitutedproduct and/or another type of 1-substituted derivative than the1-thioethyl substituted product, can be obtained by instead of(6-OBn)GlcNH₂ βSEt employing another 6-O- and/or 1-substituted acceptoras exemplified in the description.

EXAMPLE 3

Synthesis of Galβ1-3GlcNH₂ βSEt and Galβ1-3GalNH₂ βSEt. See example 1,similar conditions and enzyme may be used, but instead GlcNH₂ βSEt, orGalNH₂ βSEt to obtain the latter product, is used as the acceptor. Here,extraction is less favorable for isolation, and instead ion-exchanger asabove may be used followed by e.g. precipitation or a secondchromatographic step.

EXAMPLE 4

Synthesis of Galβ1-4GlcNH₂ βSEt. See example 3 for acceptor substrateand isolation. Here, the source of enzyme is used which gives the 1-4linked product (cf. example 2). If a microorganism like in example 2above is used then a fermentation like in example 2 may be used.

EXAMPLE 5

Synthesis of Galβ1-3(6-OAll)GlcNH₂ βSEt. This compound and other6-substituted derivatives and other 1-substituted derivatives isobtained as in example 1 above, but instead of the 6-O-bensylaminosaccharide the 6-O-allyl- or another 6-substituted derivativeand/or another type of 1-substituted derivative is used as acceptor asmentioned in the description.

EXAMPLE 6

Synthesis of Galβ1-4(6-OAll)GlcNH₂ βSEt. This compound and other6-substituted derivatives and 1-substituted derivatives is obtained asin example 2 above employing a β-galactosidase which gives a 1-4-linkedproduct, but instead of the 6-O-bensyl aminosaccharide the 6-O-allyl- oranother 6-substituted derivative or another type of 1-substitutedderivative is used as acceptor as mentioned in the description.

EXAMPLE 7

Synthesis of Galβ1-3(4-OBn)GlcNH₂ βSEt. This compound and other4-substituted derivatives and other 1-substituted derivatives isobtained as in example 1 above employing an enzyme which gives a1-3-linked product, but instead of the 6-O-bensyl aminosaccharide the4-O-bensyl- or another 4-substituted derivative and/or 1-substitutedderivative is used as acceptor as mentioned in the description.

EXAMPLE 8

Synthesis of Galβ1-4(3-OBn)GlcNH₂ βSEt. This compound and other3-substituted derivatives is obtained as in example 2 above employing aβ-galactosidase which gives a 1-4-linked product, but instead of the6-O-bensyl aminosaccharide the 3-O-bensyl- or another 3-substitutedderivative is used as acceptor.

EXAMPLE 9

Synthesis of Fucα1-4(6-OBn)GlcNH₂ βSEt. The reaction was carried out atroom temperature with initial concentration of substrates typically inthe range 0.06 M to 0.1 M. A crude ammonium sulphate precipitate of theenzyme was used in the reaction, which was carried out at pH 5 in 0.05 Msodium acetate buffer. The reaction was terminated by heat treatment forca 5 minutes in a boiling water-bath. The product was isolated by e.g.adjusting the pH to ca 10.5 (minimizing the charge on the amino-group),extraction of the water phase with ethyl acetate, followed by butanolextraction, the butanol phase was evaporated and the residue dissolvedin water and applied to an ion-exchanger (in this example a sulphopropylgroup containing fast-flow ion-exchanger from Pharmacia). The fractionscontaining the product was evaporated and the product dried and analyzedby NMR.

Similarly, another 6-O-substituted product than the bensyl-substitutedproduct and/or another type of 1-substituted derivative than the1-thioethyl substituted product, can be obtained by instead of(3-OBn)GlcNH₂ βSEt employing another 6-O- and/or 1-substituted acceptoras exemplified in the description.

EXAMPLE 10

Synthesis of Fucα1-3(6-OBn)GlcNH₂ βSEt. The synthesis of this compoundis achieved similarly as above, but another source of enzyme, whichgives the α1-3 linked product, is employed. Similarly, another6-O-substituted product than the bensyl-substituted and/or another typeof 1-substituted derivative than the 1-thioethyl substituted product,can be obtained by instead of (6-OBn)GlcNH₂ βSEt employing another 6-O-and/or 1-substituted acceptor.

EXAMPLE 11

Synthesis of Fucα1-3(4-OBn)GlcNH₂ βSEt. The synthesis of this compoundis achieved similarly as above, but with (4-OBn)GlcNH₂ βSEt as theacceptor. Similarly, another 4-O-substituted product than thebensyl-substituted and/or another type of 1-substituted derivative thanthe 1-thioethyl substituted product, can be obtained by instead of(4-OBn)GlcNH₂ βSEt employing another 4-0- and/or 1-substituted acceptor.

EXAMPLE 12

Synthesis of Fucα1-4(3-OBn)GlcNH₂ βSEt. The synthesis of this compoundis achieved similarly as above, but with an enzyme which gives theα1-4-linked product and with (3-OBn)GlcNH₂ βSEt as the acceptor.Similarly, another 3-0-substituted product than the bensyl-substitutedand/or another type of 1-substituted derivative than the 1-thioethylsubstituted product, can be obtained by instead of (3-OBn)GlcNH₂ βSEtemploying another 3-O- and/or 1-substituted acceptor.

EXAMPLE 13

Synthesis of compounds of the type GlcNAcβ1-3(6-OBn)GlcNH₂ βSEt,GlcNAcβ1-4(6-OBn)GlcNH₂ βSEt, GlcNAcβ1-4(3-OBn)GlcNH₂ βSEt,GlcNAcβ1-3(4-OBn)GlcNH₂ βSEt, GlcNAcβ1-3(6-OBn)GalNH₂ βSEt,GlcNAcβ1-4(6-OBn)GalNH₂ βSEt, GlcNAcβ1-4(3-OBn)GalNH₂,βSEt andGlcNAcβ1-3(4-OBn)GalNH₂ βSEt as well as other amino-saccharides of theabove type substituted in the 1, 3, 4, or 6-positions with other type ofgroups, including saccharides, mentioned in the description, areobtained by using N-acetyl-β-D-glucosaminidase which gives the desiredlinkage, and by using as acceptor the proper one of (6-OBn)GlcNH₂ βSEt,(3-OBn)GlcNH₂ βSEt, (4-OBn)GlcNH₂ βSEt, (6-OBn)GalNH₂ βSEt,(3-OBn)GalNH₂ βSEt and (4-OBn)GalNH₂ βSEt as well as otheramino-saccharides of the above type substituted in the 1, 3, 4, or6-positions with other type of groups, including saccharides, mentionedin the description. As glycosyl donor one can use GlcNAc, a glycosidethereof such as the F-β-glycoside or the nitrophenyl-β-glycoside.

EXAMPLE 14

Synthesis of compounds of the type GalNAcβ1-3(6-OBn)GlcNH₂ βSEt,GalNAcβ1-4(6-OBn)GlcNH₂ βSEt, GalNAcβ1-4(3-OBn)GlcNH₂ βSEt,GalNAcβ1-3(4-OBn)GlcNH₂ βSEt, GalNAcβ1-3(6-OBn)GalNH₂ βSEt,GalNAcβ1-4(6-OBn)GalNH₂ βSEt, GalNAcβ1-4(3-OBn)GalNH₂ βSEt andGalNAcβ1-3(4-OBn)GalNH₂ βSEt as well as other amino-saccharides of theabove type substituted in the 1, 3, 4, or 6-positions with other type ofgroups, including saccharides, mentioned in the description, areobtained by using N-acetyl-β-D-galactosaminidase or another properβ-hexosaminidase which gives the desired linkage, and by using asacceptor the proper one of (6-OBn)GlcNH₂ βSEt, (3-OBn)GlcNH₂ βSEt,(4-OBn)GlcNH₂ βSEt, (6-OBn)GalNH₂ βSEt, (3-OBn)GalNH₂ βSEt and(4-OBn)GlcNH₂ βSEt as well as other amino-saccharides of the above typesubstituted in the 1, 3, 4, or 6-positions with other type of groups,including saccharides, mentioned in the description. As glycosyl donorone can use GalNAc, a glycoside thereof such as the F-β-glycoside or thenitrophenyl-β-glycoside.

EXAMPLE 15

Synthesis of compounds of the type GalNAcα1-3(6-OBn)GlcNH₂ βSEt,GalNAcα1-4(6-OBn)GlcNH₂ βSEt, GalNAcα1-4(3-OBn)GlcNH₂ βSEt,GalNAcα1-3(4-OBn)GlcNH₂ βSEt, GalNAcα1-3(6-OBn)GalNH₂ βSEt,GalNAcα1-4(6-OBn)GalNH₂ βSEt, GalNAcα1-4(3-OBn)GalNH₂ βSEt andGalNAcα1-3(4-OBn)GalNH₂ βSEt as well as other amino-saccharides of theabove type substituted in the 1, 3, 4, or 6-positions with other type ofgroups, including saccharides, mentioned in the description, areobtained by using N-acetyl-α-D-galactosaminidase or another properα-hexosaminidase which gives the desired linkage, and by using asacceptor the proper one of (6-OBn)GlcNH₂ βSEt, (3-OBn)GlcNH₂ βSEt,(4-OBn)GlcNH₂ βSEt, (6-OBn)GalNH₂ βSEt, (3-OBn)GalNH₂ βSEt and(4-OBn)GlcNH₂ βSEt as well as other amino-saccharides of the above typesubstituted in the 1, 3, 4, or 6-positions with other type of groups,including saccharides, mentioned in the description. As glycosyl donorone can use GalNAc, a glycoside thereof such the F-β-glycoside ornitrophenyl-β-glycoside.

EXAMPLE 16

Synthesis of compounds of the type Manα1-3(6-OBn)GlcNH₂ βSEt,Manα1-4(6-OBn)GlcNH₂ βSEt, Manα1-4(3 OBn)GlcNH₂ βSEt,Manα1-3(4-OBn)GlcNH₂ βSEt, Manα1-3(6-OBn)GalNH₂ βSEt,Manα1-4(6-OBn)GalNH₂ βSEt, Manα1-4(3-OBn)GalNH₂ βSEt andManα1-3(4-OBn)GalNH₂ βSEt as well as other amino-saccharides of theabove type substituted in the 1, 3, 4, or 6-positions with other type ofgroups, including saccharides, mentioned in the description, areobtained by using α-D-mannosidase which gives the desired linkage, andby using as acceptor the proper one of (6-OBn)GlcNH₂ βSEt, (3-OBn)GlcNH₂βSEt, (1-OBn)GlcNH₂ βSEt, (6-OBn)GalNH₂ βSEt, (3-OBn)GalNH₂ βSEt and(4-OBn)GlcNH₂ βSEt as well as other amino-saccharides of the above typesubstituted in the 1, 3, 4, or 6-positions with other type of groups,including saccharides, mentioned in the description. As glycosyl donorone can use mannose, a glycoside thereof such as the F-β-glycoside orthe nitrophenyl-β-glycoside.

EXAMPLE 17

Synthesis of compounds of the type Glcβ1-3(6-OBn)GlcNH₂ βSEt,Glcβ1-4(6-OBn)GlcNH₂ βSEt, Glcβ1-4 (3-OBn)GlcNH₂ βSEt,Glcβ1-3(4-OBn)GlcNH₂ βSEt, Glcβ1-3(6-OBn)GalNH₂ βSEt,Glcβ1-4(6-OBn)GalNH₂ βSEt, Glcβ1-4(3-OBn)GalNH₂ βSEt andGlcβ1-3(4-OBn)GalNH₂ βSEt as well as other amino-saccharides of theabove type substituted in the 1, 3, 4, or 6-positions with other type ofgroups, including saccharides, mentioned in the description, areobtained by using β-D-glucosidase which gives the desired linkage and byusing as acceptor the proper one of (6-OBn)GlcNH₂ βSEt, (3-OBn)GlcNH₂βSEt, (4-OBn)GlcNH₂ βSEt, (6-OBn)GalNH₂ βSEt, (3-OBn)GalNH₂ βSEt and(4-OBn)GlcNH₂ βSEt as well as other amino-saccharides of the above typesubstituted in the 1, 3, 4, or 6-positions with other type of groups,including saccharides, mentioned in the description. As glycosyl donorone can use mannose, a glycoside thereof such as the F-β-glycoside orthe nitrophenyl-β-glycoside.

In examples 13, 14, 15, 16 and 17 above similar isolation procedures asin example 1 may be used.

Other saccharides than those mentioned above are obtained by using otherglycosidases, including α- or β-xylosidases, α-sialidases andendoglycosidases, and other glycosyl donors as mentioned in thedescription.

A few non-limiting examples of the use of the invention for preparationof amino-deoxy-containing trisaccharides and higher saccharides inconjunction with glycosyltransferases are given below. Theglycosyltransferases may be used in more or less isolated form, and maybe of natural origin or may be obtained by any recombinant techniques.The glycosyl donors for the glycosyl transferases may be nucleotidesugars or modified nucleotide sugars or any type of glycosyl donor whichcan be used to promote the glycosyltranferase reaction. It is well knownthat glycosyltransferases can transfer modified and unnatural glycosylunits and di- tri- and higher oligosaccharides to their acceptors andthis can also be used in the invention.

Moreover the glycosyl donors for the glycosyltransferase reactions canbe produced either separately or in situ in the reaction vessel (by forinstance multi-enzyme systems) and this does not limit the scope of theinvention. Also, the glycosidase reaction can be either carried outseparately or in the same reaction vessel as the glycosyltransferasereaction and this does not limit the scope of the invention. Moreover,either or both of the glycosidase and the glycosyltransferase can beused in soluble form or in immobilized form to any of the materialsmentioned in the description.

EXAMPLE 18

Synthesis of NeuAcα2-3Galβ1-3GlcNH₂ βSEt. Galβ1-3GlcNH₂ βSEt is preparedas described above and used directly or after isolation as acceptor fora β-D-galactoside α2-3-sialyltransferase (e.g. EC 2.4.99.4) reactionwith a suitable glycosyl donor such as CMP-NeuAc. Similarly, another1-substituted product than the 1-thioethyl substituted product above canbe obtained by instead of GlcNH₂ βSEt employing another type of1-substituted acceptor as exemplified in the description.

EXAMPLE 19

Synthesis of NeuAcα2-3Galβ1-4GlcNH₂ βSEt, Galβ1-1GlcNH₂ βSEt is preparedas described above and used directly or after isolation as acceptor fora β-D-galactoside α2-3-sialyltransferase.(e.g. EC 2.4.99.5) reactionwith a suitable glycosyl donor such as CMP-NeuAc. Similarly, another1-substituted product than the 1-thioethyl substituted product above,can be obtained by instead of GlcNH₂ βSEt employing another type of1-substituted acceptor as exemplified in the description.

EXAMPLE 20

Synthesis of NeuAcα2-3Galβ1-4(6-OBn)GlcNH₂ βSEt. Galβ1-4(6-OBn)GlcNH₂βSEt is prepared as described above and used directly or after isolationas acceptor for a β-D-galactoside α2-3-sialyltranferase (e.g. EC2.4.99.5) reaction with a suitable glycosyl donor such as CMP-NeuAc.Similarly, another 6- and/or 1-substituted product than the 6-O-bensyland 1-thioethyl substituted product above can be obtained by instead of6-O-bensyl-GlcNH₂ βSEt employing another type of 6- and/or 1-substitutedacceptor as exemplified in the description.

EXAMPLE 21

Synthesis of NeuAcα2-3Galβ1-3(4-OBn)GlcNH₂ βSEt. Galβ1-3(4-OBn)GlcNH₂βSEt is prepared as described above and used directly or after isolationas acceptor for a β-D-galactoside α2-3-sialyltransferase (e.g. EC2.4.99.4) reaction with a suitable glycosyl donor such as CMP-NeuAc.Similarly, another 4- and/or 1-substituted product than the 4-O-bensyland 1-thioethyl substituted product above, can be obtained by instead of4-O-bensyl-GlcNH₂ βSEt employing another type of 4- and/or 1-substitutedacceptor as exemplified in the description.

EXAMPLE 22

Synthesis of NeuAcα2-3Galβ1-4(3-OBn)GlcNH₂ βSEt. Galβ1-4(3-OBn)GlcNH₂βSEt is prepared as described above and used directly or after isolationas acceptor for a β-D-galactoside α2-3-sialyltransferase (e.g. EC2.4.99.5) reaction with a suitable glycosyl donor such as CMP-NeuAc.Similarly, another 3- and/or 1-substituted product than the 3-O-bensyland 1-thioethyl substituted product above, can be obtained by instead of3-O-bensyl-GlcNH₂ βSEt employing another type if 3- and/or 1-substitutedacceptor as exemplified in the description.

EXAMPLE 23

Synthesis of NeuAcα2-6Galβ1-4GlcNH₂ βSEt. Galβ1-4GlcNH₂ βSEt is preparedas described above and used directly or after isolation as acceptor fora β-D-galactoside α2-6-sialyltransferase (e.g. EC 2.4.99.1) reactionwith a suitable glycosyl donor such as CMP-NeuAc.

EXAMPLE 24

Synthesis of Galα1-3Galβ1-4GlcNH₂ βSEt. Galβ1-4GlcNH₂ βSEt is preparedas described above and used directly or after isolation as acceptor fora α1-3-D-galactosyltransferase (e.g. EC 2.4.1 151) reaction with asuitable glycosyl donor such as UDP-Gal.

EXAMPLE 25

Synthesis of Galβ1-4(Fucα1-3)GlcNH₂ βSEt. Galβ1-4GlcNH₂ βSEt is preparedas described above and used directly or after isolation as acceptor fora α1-3-fucosyltransferase (e.g. EC 2.4.1.152 or 65) reaction with asuitable glycosyl donor such as GDP-Fuc.

EXAMPLE 26

Synthesis of Fucα1-2Galβ1-4GlcNH₂ βSEt. Galβ1-4GlcNH₂ βSEt is preparedas described above and used directly or after isolation as acceptor fora α1-2-fucosyltransferase (e.g. EC 2.4.1.69) reaction with a suitableglycosyl donor such as GDP-Fuc.

EXAMPLE 27

Synthesis of Fucα1-2Galβ1-3GlcNH₂ βSEt. Galβ1-3GlcNH₂ βSEt is preparedas described above and used directly or after isolation as acceptor fora α1-2-fucosyltransferase (e.g. EC 2.4.1.69) reaction with a suitableglycosyl donor such as GDP-Fuc.

EXAMPLE 28

Synthesis of NeuAcα2-3Galβ1-3GalNH₂ βSEt. Galβ1-3GalNH₂ βSEt is preparedas described above and used directly or after isolation as acceptor fora α2-3-sialyltransferase (e.g. EC 2.4.99.4) reaction with a suitableglycosyl donor such as CMP-NeuAc.

EXAMPLE 29

Synthesis of NeuAcα2-3Galβ1-3(NeuAcα2-6)GalNH₂ βSEt.NeuAcα2-3Galβ1-3GalNH₂ βSEt is prepared as described above and useddirectly or after isolation as acceptor for a α2-6-sialyltransferase(e.g. EC 2.4.99.7) reaction with a suitable glycosyl donor such asCMP-NeuAc.

In the examples 23 to 29 above, other 1-substituted products than the1-thioethyl substituted products above, can be obtained by instead ofGlcNH₂ βSEt employing another type of 1-substituted acceptor asexemplified in the description.

In the isolation of the compounds above, precipitation from water may beused if hydrophobic groups are present on the acceptors. Also extractionof the product from a solid crude mixture may be used with a suitablesolvent e.g. MeOH. These techniques, precipitation and extraction arecomplementary to chromatography and a combination of one, two or allthree of these techniques may be used for isolation.

Further variations and modifications of the foregoing will be apparentto those skilled in the art and such variations and modifications areattended to be encompassed by the claims that are appended hereto.

Swedish Priority Application 9301677-2 filed on May 17, 1993 is reliedon and incorporated by reference.

U.S. Pat. No. 5,246,840; U.S. Pat. No. 4,918,009; U.S. Pat. No.4,415,665; U.S. patent application Ser. No. 07/834,575, filed on Feb.18, 1992, now U.S. Pat. No. 5,372,937; and U.S. patent application Ser.No. 07/940,866, filed on Oct. 29, 1992, now abandoned are incorporatedby reference in their entirety (especially for their teachingsconcerning acceptor substances, donor substances, and enzymes). WO93/03168 (PCT/SE92/00541) is incorporated by reference in its entirety(especially for its teachings concerning acceptor substances, donorsubstances, and enzymes).

I claim:
 1. An amino-deoxy di- or olisosaccharide compound which isselected from the group consisting of Galβ1-3(6-O-Bn)GlcNH₂ βSEt,Fucα1-4 (6-O-Bn)GlcNH₂ βSEt, Gal1-4(6-OBn)GlcNH₂ SEt, Galβ1-3GlcNH₂βSEt, Galβ1-3GalNH₂ βSEt, Galβ1-4GlcNH₂ βSEt, Galβ1-3(6-OAll)GlcNH₂βSEt, Galβ1-4(6-OAll)GlcNH₂ βSEt, Galβ1-3(4-OBn)GlcNH₂ βSEt,Galβ1-4(3-OBn)GlcNH₂ βSEt, Fucα1-4(6-OBn)GlcNH₂ βSEt,Fucα1-3(6-OBn)GlcNH₂ βSEt, Fucα1-3(4-OBn)GlcNH₂ βSEt,Fucα1-4(3-OBn)GlcNH₂ βSEt, GlcNAcβ1-3(6-OBn)GlcNH₂ βSEt,GlcNAcβ1-4(6-OBn)GlcNH₂ βSEt, GlcNAcβ1-4(3-OBn)GlcNH₂ βSEt,GlcNAcβ1-3(4-OBn)GlcNH₂ βSEt, GlcNacβ1-3(6-OBn)GalNH₂ βSEt,GlcNacβ1-4(6-OBn)GalNH₂ βSEt, GlcNacβ1-4(3-OBn)GalNH₂ βSEt,GlcNAcβ1-3(4-OBn)GalNH₂ βSEt, GalNAcβ1-3(6-OBn)GlcNH₂ βSEt,GalNAcβ1-4(6-OBn)GlcNH₂ βSEt, GalNAcβ1-4(3-OBn)GlcNH₂ βSEt,GalNAcβ1-3(4-OBn)GlcNH₂ βSEt, GalNAcβ1-3(6-OBn)GalNH₂ βSEt,GalNAcβ1-4(6-OBn)GalNH₂ βSEt, GalNAcβ1-4(3-OBn)GalNH₂ βSEt,GalNAcβ1-3(4-OBn)GalNH₂ βSEt, GalNAcα1-3(6-OBn)GlcNH₂ βSEt,GalNAcα1-4(6-OBn)GlcNH₂ βSEt, GalNAcα1-4(3-OBn)GlcNH₂ βSEt,GalNAcα1-3(4-OBn)GlcNH₂ βSEt, GalNAcα1-3(6-OBn)GalNH₂ βSEt,GalNAcα1-4(6-OBn)GalNH₂ βSEt, GalNAcα1-4(3-OBn)GalNH₂ βSEt,GalNAcα1-3(4-OBn)GalNH₂ βSEt, Manα1-3(6-OBn)GlcNH₂ βSEt,Manα1-4(6-OBn)GlcNH₂ βSEt, Manα1-4(3-OBn)GlcNH₂ βSEt,Manα1-3(4-OBn)GlcNH₂ βSEt, Manα1-3(6-OBn)GalNH₂ βSEt,Manα1-4(6-OBn)GalNH₂ βSEt, Manα1-4(3-OBn)GalNH₂ βSEt,Manα1-3(4-OBn)GalNH₂ βSEt, Glcβ1-3(6-OBn)GlcNH₂ βSEt,Glcβ1-4(6-OBn)GlcNH₂ βSEt, Glcβ1-4(3-OBn)GlcNH₂ βSEt,Glcβ1-3(4-OBn)GlcNH₂ βSEt, Glcβ1-3(6-OBn)GalNH₂ βSEt,Glcβ1-4(6-OBn)GalNH₂ βSEt, Glcβ1-4(3-OBn)GalNH₂ βSEt,Glcβ1-3(4-OBn)GalNH₂ βSEt, NeuAcα2-3Galβ1-3GlcNH₂ βSEt,NeuAcα2-3Galβ1-4GlcNH₂ βSEt, NeuAcα2-3Galβ1-4(6-OBn)GlcNH₂ βSEt,NeuAcα2-3Galβ1-3(4-OBn)GlcNH₂ βSEt, NeuAcα2-3Galβ1-4(3-OBn)GlcNH₂ βSEt,NeuAcα2-6Gal1-4GlcNH₂ βSEt, Galα1-3Galβ1-4GlcNH₂ βSEt,Galβ1-4(Fucα1-3)GlcNH₂ βSEt, Fucα1-2Galβ1-4GlcNH₂ βSEt,Fucα1-2Galβ1-3GlcNH₂ βSEt, NeuAcα2-3Galβ1-3GalNH₂ βSEt, andNeuAcα2-3Galβ1-3(NeuAcα2-6)GalNH₂ βSEt.